The objective of this First proposal is to elucidate the role of Nef in HIV pathogenesis. A combined molecular genetic, biochemical, and virologic approach will be used to de fine the cellular interactions and biologic function of Nef and to ascertain whether these properties are critical for viral pathogenesis. Hypothesis; The interaction between the Nefs of HIV and SIV and cellular factors, including a newly recognized protein kinase, are critical for the maintenance of high viral loads and disease progression. Specific Aim 1: To elucidate the mechanism of Nef action, it is important to identify the cellular proteins that interact with Nef. Affinity chromatography will be used to identify and characterize the cellular proteins that associate with Nef; emphasis will be directed towards determining the identity and properties of the Nef-associated protein kinase . Specific Aim 2: To define the functional domain(s) of Nef and identify critical determinants for the interactions between Nef and cellular proteins; site-specific Nef mutants will be constructed and tested for kinase association, enhancement of viral infectivity, T cell activation, and CD4 down-regulation. Specific Aim 3: To define the role(s) of subcellular localization and membrane targeting in determining the interactions between Nef and its intracellular targets, and to determine whether the partitioning of Nef into specific subcellular compartments affects its phenotypic properties. Specific Aim 4: To determine whether the interaction of Nef with the cellular protein kinase activity is essential for viral persistence and progression to AIDS in vivo, by using SIV infection of rhesus macaques. Significance: The global spread of HIV infection and AIDS continues while current therapies are very limited and effective vaccines are nonexistent. Accordingly, novel therapeutic approaches must be devised to target viral gene functions that regulate virus replication nd produce disease. Thus, knowledge of Nef function will not only aid in elucidating the mechanism of viral pathogenesis, but also may be used to design novel therapies that inhibit virus replication and block progression to AIDS.